(A) REMSA shows binding of miR-328, CEBPA uORF, and miR-330 RNA probes to MBP-tagged hnRNP E2 (lanes 2, 4, 6, 7–12), MBP (lanes 1, 3, 5), and cytoplasmic lysates of 32Dcl3 (lanes 14, 19) and untreated or imatinib-treated 32D-BCR/ABL cells (lanes 13, 15–18, 20–22). Cold competitor RNAs were used as indicated (lanes 8–9, 11–12, 17–18, 22).
(B) Top: UV crosslinking shows binding of miR-328 (lanes 1–3 and 10), CEBPA uORF (lanes 4–6), the seed sequence-mutated miR-328 (miR-328-Mut) (lanes 7–9), and miR-330 (lane 11) to hnRNP E2 in 32Dcl3, 32D-BCR/ABL, and 32D-Flag-E2 cell lysates (lanes 1–6). Bottom: Western Blots show levels of hnRNP E2 and GRB2 in the lysates used in UV crosslinking. Sequence of the wild-type and seed sequence-mutated miR-328 with substituted nucleotides indicated by asterisks.
(C) RNA Immunoprecipitation (RIP) assay for miR-328 performed on anti-hnRNP E2 immunoprecipitates (IPs) from lysates of untreated (lane 3), imatinib-treated (lane 5), and pSR-miR-328-transduced (lane 7) 32D-BCR/ABL (6.15 clone) cells. RIP with a nonrelated IgG served as controls (lane 8). IN: input RNA; MWM: molecular weight marker (lane 1); NTC: nontemplate control;-RT: no reverse transcribed PCR reaction. RIP was also observed with ectopically expressed Flag-hnRNP E2 proteins (see Figure S1).
(D) Left: RIP assays for CEBPA mRNA (top) and miR-328 (bottom) performed on anti-hnRNP E2 (lanes 5 and 9) and nonrelated IgG (lanes 3 and 7) IPs from parental (lanes 2–5) and miR-328-expressing (lanes 6–9) 32D-BCR/ABL cells. IN: RNA input; MWM: molecular weight marker (lane 1); NTC: nontemplate PCR control. hnRNP E2 RIP assays for a nonrelated mRNA (i.e., SET) are reported in Figure S1. Inset top right: Northern blot shows levels of ectopic miR-328 in vector- (lanes 1 and 3) and miR-328-transduced (lanes 2 and 4) 32Dcl3 and 32D-BCR/ABL cells. snRNA U6 was analyzed for normalization. Right: Densitometric analyses of the levels of CEBPA mRNA and miR-328 associated to hnRNP E2 evaluated by RIP assays (n = 4) performed with parental (light bars) and miR-328-expressing (dark bars) 32D-BCR/ABL cells (mean ± SEM).
(E) Left: RIP assays for CEBPA mRNA (top) on anti-hnRNP E2 IPs from vector (pSuper)- (lane 5), miR-328- (lane 7), and miR-181b-transduced (lane 9) 32D-BCR/ABL cells; RIP assays for miR-181b (middle) and miR-328 (bottom) on anti-hnRNP E2 IPs from miR-181b-expressing 32D-BCR/ABL cells (lanes 8 and 9). Inset top right: UV crosslinking with miR-328, miR-181b, and miR-330 ³²P-labeled oligoribonucleotides and cytoplasmic lysates of 32Dcl3 (lanes 1, 3, and 5) and 32D-BCR/ABL (lanes 2, 4, and 6) cells. Binding of hnRNP E2 to miR-328 is shown in lane 2. Inset bottom left: Graph shows qRT-PCR analysis of miR-181b expression in 32Dcl3 (black), vector- (gray), and pSUP-miR-181b-transduced (white) 32D-BCR/ABL cells (mean ± SEM). Right: Densitometric analysis of the RIP’ed CEBPA mRNA associated to hnRNP E2 (n = 3), expressed as percentage of the input RNA (IN), in vector-, miR-328-, and miR-181b-transduced 32D-BCR/ABL cells (mean ± SEM). Controls for endogenous and ectopic hnRNP E2 protein and miR-328 expression levels and the specificity of the RIP protocol are shown in Figure S1.

(A) miR-328 levels in (left) 32Dcl3 cells undergoing G-CSF-induced differentiation; (middle) Lin⁻/Sca⁺/Kit⁺ HSC, CMP/GMP/MEP committed progenitors and mature neutrophil BM subpopulations from wild-type C57BL/6 mice (mean ± SEM); and (right) CD34⁺ human BM cells undifferentiated (white) and induced to differentiate for the indicated time toward the erythroid (light gray), megakaryocytic (dark gray), granulocytic (red), or monocytic (black) lineages (mean ± SEM).
(B) Wright-Giemsa-stained cytospins of G-CSF-treated (0–7 days) pSuper-, miR-328-, miR-328-Mut-, miR-223-, and/or miR-181b-infected 32Dcl3 and/or 32D-BCR/ABL cells (mean ± SEM). Levels of miR-223 in BCR/ABL⁺ cell lines and primary cells and effect of ectopic miR-223 on cell proliferation are reported in Figure S2.
(C) Wright-Giemsa-stained cytospins of primary G-CSF-treated (0–10 days) uninfected and miR-328-, miR-328-Mut-, and miR-223-infected CML-BC^CD34+ BM progenitors (mean ± SEM). For levels of ectopic miR-328 and miR-223 expression in primary CML-BC cells, see Figure S2.
(D) Myeloperoxidase (MPO) immunostaining of G-CSF-treated uninfected and miR-328- and miR-223-transduced CML-BC^CD34+ BM cells. Data are representative of three independent experiments (mean ± SEM).
(E) Western blot shows C/EBPα, hnRNP E2, and GRB2 levels in G-CSF-treated 32Dcl3 and empty vector-, miR-328-, miR-223-, miR-328-Mut-, and/or miR-181b-infected 32D-BCR/ABL and CML-BC^CD34+ BM cells (right).
(F) qRT-PCR shows levels of CEBPA in empty vector-, miR-223-, or miR-328-infected CML-BC cells (mean ± SEM). qRT-PCR showing CEBPA mRNA levels in empty vector-, miR-223-, or miR-328-transduced 32D-BCR/ABL cells (mean ± SEM) is reported in Figure S2.

miR-328 levels in 32Dcl3 and/or 32D-BCR/ABL cells (A) treated with imatinib or the MAPK inhibitors U0126 and CI-1040 or (B) expressing a Flag-hnRNP E2 (left) or a shRNA-targeting hnRNP E2 (right).
(C) Top: Representation of the C/EBPα-binding sites within the miR-328 promoter; bottom: Chromatin immunoprecipitation (ChIP) with anti-HA antibody shows binding of HA-C/EBPα to miR-328 promoter sequences in 32D-HA-C/EBPα cells (left: ChIP blot; middle: densitometric analysis). Anti-Flag immunoprecipitates served as negative controls. Bars indicate the mean ± SEM from three independent experiments; northern and western blots (right) show levels of miR-328 and HA-C/EBPα, respectively, in parental and 32D-HA-C/EBPα cells. U6 snRNA and GRB2 protein levels were used as controls.
(D) Model of the molecular network regulating miR-328 expression in CML-BC and miR-328 decoy activity in BCR/ABL⁺ myeloid cell differentiation by direct interference with hnRNP E2 translation inhibition of C/EBPα expression.

(A) hnRNP E2 consensus binding sites (red) in the precursor and mature (gray box) miR-328 and in the CEBPA uORF/spacer region.
(B) Left: Northern blot and RT-PCR show miR-328 levels and western blot shows BCR/ABL levels in 32Dcl3 and untreated or imatinib-treated 32D-BCR/ABL cells; right: northern blot and RT-PCR show miR-328 levels in vector- and BCR/ABL-infected lineage-negative (Lin⁻) mouse BM cells.
(C) qRT-PCR shows miR-328 levels in untreated (n = 3) and imatinib-treated (n = 3) Lin⁻ BM cells from leukemic SCLtTA-BCR/ABL mice (left) and in CD34⁺ BM progenitors from CML-CP (n = 6) and CML-BC (n = 6) patients (right). snRNA U6 and GRB2 levels were used as loading controls (mean ± standard error of the mean [SEM]).

Western blots show levels of Dicer, TRBP2, Ago2, and/or hnRNP E2 in cytoplasmic lysates (input; lanes 1 and 4), in anti-hnRNP E2 (A) and anti-Dicer (B) IPs (lane 3), in IPs with an isotype-matched IgG (lane 2), and in hnRNP E2-and Dicer-immunodepleted (ID) lysates (lane 5) of 32D-BCR/ABL-miR-328cells. (C) RIP assay for miR-328 on anti-Dicer (lane 4), anti-hnRNP E2 (lane 5), and anti-Ago2 (lane 7) IPs from 32D-BCR/ABL-miR-328 cell lysates. Note that the anti-hnRNP E2 RIP for miR-328 was performed on Dicer-immunodepleted lysates. RIP with a nonrelated IgG served as a negative control.

(A) Levels of newly synthesized ³⁵S-C/EBPα protein in RRL translation reactions programmed with CEBPA mRNA (derived from pcDNA3-WT-uORF-C/EBPα) (black), CEBPA mRNA and mature miR-328 RNA oligonucleotides (dark gray), CEBPA mRNA and recombinant MBP-hnRNP E2 protein either alone (light gray) or in the presence of mature miR-328 (red), or miR-330 (white; negative control) RNA oligonucleotides. Data are expressed as percentage of the mean ± SEM and are representative of three different experiments performed in duplicate. Inset: Western blot shows levels of both endogenous RRL hnRNP E2 and recombinant MBP-hnRNP E2.
(B) Wright-Giemsa-stained cytospins of G-CSF-treated (0–7 days) 6.15-pSUP, 6.15-miR-328, and 6.15-WT-uORF-miR-328 cells (mean ± SEM).
(C) Left: Levels of hnRNP E2, endogenous and HA-tagged C/EBPα, and GRB2 proteins and miR-328 and snRNA U6 in parental 32Dcl3, 6.15-pSUP-transduced, and miR-328-transduced 6.15-WT-uORF cells; right: RT-PCR and qRT-PCR show levels of CEBPA mRNA in 32Dcl3, 6.15, 6.15-miR-328, and 6.15-WT-uORF-HA-CEBPA cells either uninfected or infected with pSUP or miR-328 constructs. GAPDH levels were measured for normalization (mean ± SEM).
(D) RIP assays for CEBPA mRNA (top) and miR-328 (bottom) on anti-hnRNP E2 (lanes 5 and 9) and nonrelated IgG (lanes 3 and 7) IPs from 6.15-uORF-pSUP (lanes 2–5) and 6.15-uORF-miR-328 cells (lanes 6–9). IN: input RNA.
(E) Top: H&E-staining of BM shows maturation of BCR/ABL⁺ cells in mice injected with p-SUPERIOR vector- (middle) and miR-328-transduced (right) 6.15-WT-uORF cells. Age-matched mice (left) served as a control. FACS analysis shows mean fluorescence intensity (MFI; mean ± SEM) of differentiated GFP⁺Gr1⁺BCR/ABL⁺ cells at 3 weeks post-transplant from BM of 3 mice/group. Bottom: Visual analysis and weight of spleens from the same groups of mice (mean ± SEM).

(A) IL-3-independent or -dependent methylcellulose colony formation (mean ± SEM from triplicates of three independent experiments) of vector- (gray bars) and miR-328-transduced (red bars) 32D-BCR/ABL cells (IL-3-independent), leukemic Lin⁻ SCLtTA-BCR/ABL (n = 5) (IL-3-dependent) cells, CML-BC^CD34+ (n = 2) (IL-3 dependent) BM progenitors, and vector- (pSUPER) or hnRNP E2 shRNA (pSUPER-shE2)-infected 32D-BCR/ABL cells (IL-3-independent). Inset: Western blot shows hnRNP E2 levels upon shRNA knockdown.
(B) miR-328-binding site (red) within the 3′UTR of mouse and human PIM1 mRNA.
(C) PIM1 protein levels in 32Dcl3, untreated or imatinib-treated 32D-BCR/ABL (wild-type and T315I) and K562 cells (left); in CD34⁺ BM cells from healthy donors (NBM), CML-CP, and CML-BC patients (middle); and in the CD34⁻, untreated and imatinib-treated CD34⁺ BM fractions from a CML-BC patient.
(D) Left: Effect of miR-328 expression on PIM1 protein levels in 32D-BCR/ABL, K562, and CML-BC^CD34+ BM cells. 32Dcl3 cells were used as a negative control; right: PIM1 mRNA expression by qRT-PCR in 32Dcl3, vector-transduced, and miR-328-transduced 32D-BCR/ABL. Representative of triplicates from three independent experiments (mean ± SEM).
(E) Left: Levels of ectopic Flag-PIM1 proteins from constructs lacking (pMX-Flag-WTPIM1) and harboring the wild-type (pMX-Flag-WTPIM1-WT3′UTR) or 196 base pair end-terminal-deleted (pMX-Flag-WTPIM1-Δ3′UTR) PIM1 3′UTR in miR-223- or miR-328-expressing 32D-BCR/ABL. Northern blot shows levels of miR-223, miR-328, and snRNA U6. Right: Schematic representation of the Flag-PIM1 constructs.
(F) Effect of seed sequence-mutated (miR-328-Mut) on endogenous PIM1 expression in parental 32Dcl3 and empty vector (pCDH)-, miR-328-, and miR-328-Mut-infected 32D-BCR/ABL cells.
(G) Left: Endogenous and ectopic (wild-type [WT PIM-1] and kinase-deficient [KD PIM-1]) PIM1 protein levels in 32Dcl3, pSUPER- and pSR-miR-328-infected 32D-BCR/ABL cells; right: graph shows rescue of IL-3-independent clonogenic activity of miR-328-expressing (white) 32D-BCR/ABL cells to normal levels (black) by ectopic wild-type (red) but not kinase-deficient (yellow) PIM1 construct lacking the 3′UTR. Effect of PIM1 forced expression on vector-transduced clonogenicity (gray). Bars represent the mean ± SEM of colony numbers from three independent experiments.